Familial Mediterranean fever (FMF) is a recessively inherited disorder characterized by self-limited attacks of fever with serosal, synovial, or cutaneous inflammation, sometimes complicated by systemic amyloidosis. In 1992 our laboratory mapped the FMF locus to chromosome 16p13.3, and in 1997 we isolated the underlying gene, MEFV, and demonstrated that it is highly expressed in granulocytes. During the 6 years leading up to the present reporting period, we have focused on several areas, including FMF population genetics, the regulation of FMF gene expression in leukocyte subpopulations, the biochemistry and cell biology of pyrin (the FMF protein), and the development of animal models of FMF. During the year directly antecedent to the present reporting period, the laboratory focused on two major projects. The major findings of the first project, a study of a mouse strain expressing a truncated form of pyrin, indicated that pyrin plays a major role in the regulation of caspase-1 activation and IL-1beta processing, and that pyrin regulates macrophage apoptosis through a caspase-8-dependent, IL-1beta-independent pathway. The second project, an analysis of proteins that interact with pyrin, established that the cytoskeletal protein PSTPIP1 binds pyrin, and that PSTPIP1 mutations associated with the syndrome of pyogenic arthritis with pyoderma gangrenosum and acne (PAPA) lead to markedly increased pyrin-binding and IL-1beta activation. Results of the Last Year A major focus of the last year has been the study of the direct interaction of pyrin with caspase-1, and its potential implications in normal biology and in FMF. In a series of experiments that had begun during the FY03 reporting period, we demonstrated that antibodies to caspase-1 coimmunoprecipitate endogenous pyrin in THP-1 human monocytic cells. Conversely, we found that polyclonal antipyrin antibodies specifically precipitate caspase-1, but not caspases 2-10, from THP-1 lysates. To rule out any possible role of the ASC protein (apoptosis-associated specklike protein with a caspase recruitment domain), which can bind both pyrin and caspase-1, GST-pulldown experiments were also performed in PT67 cells, which do not express ASC even by sensitive RT-PCR. Further GST-pulldowns using deletion mutants demonstrated that the C-terminal B30.2 domain of pyrin and the catalytic p20 and p10 domains of caspase-1 are the major interaction domains. To study the biochemical consequences of this interaction, radiolabeled pyrin was incubated with purified caspase-1. In these assays we observed a time- and dose-dependent cleavage of pyrin from its 95 kDa native form to an approximately 55 kDa cleavage fragment. The cleavage of pyrin was blocked by a specific inhibitor of caspase-1 in a dose-dependent fashion. The actual cleavage site was determined by Edman sequencing, and was found to be located between Asp330 and Ser331 in pyrin. Site-directed mutagenesis of Asp330 completely abolished the caspase-1-mediated cleavage of pyrin. We next examined the subcellular localization of GFP-tagged pyrin and its N-terminal (aa 1-330) and C-terminal (aa 331-781) cleavage fragments. Whereas full-length pyrin and the C-terminal fragment were restricted to the cytoplasm, the N-terminal fragment was found in both the nucleus and the cytoplasm. Moreover, in luciferase reporter assays, the N-terminal pyrin cleavage fragment had dramatic NF-kappaB activating effects, relative to the modest stimulation seen with full-length pyrin. The NF-kappaB stimulatory activity of both full-length pyrin and the N-terminal fragment was dependent on the presence of ASC, and inactivating mutations in the PYRIN domains of either pyrin or ASC abrogated NF-kappa B activation. Finally, we examined the effect of FMF-associated mutations in pyrin?s C-terminal B30.2 domain on caspase-1 interaction, on caspase-1-mediated cleavage, and on NF-kappaB activation. Disease-associated B30.2 mutants associated less well than wild type with caspase-1, but were cleaved more efficiently. When FMF-associated pyrin mutants were cotransfected with caspase-1 in a luciferase reporter assay, there as an approximately 10-fold increase in NF-kappaB activation, relative to wild type protein. Moreover, absolute and relative quantities of cleaved pyrin were substantially increased in leukocytes from FMF patients, compared with healthy controls. Conclusions and Significance These data establish a novel inflammatory pathway in which the cleavage of pyrin by caspase-1 induces NF-kappaB activation. The heightened susceptibility of FMF-associated pyrin mutants to cleavage may help account for the autoinflammatory phenotype of FMF, and suggests a possible mechanism whereby carriers of even a single MEFV mutation sometimes exhibit clinical or biochemical evidence of episodic inflammation. Such heterozygote effects may also confer increased resistance to bacterial pathogens, possibly accounting for the high FMF carrier frequencies in certain populations. The biochemical mechanism by which N-terminal pyrin induces NF-kappaB activation is unknown, but our data renew interest in pyrin as a possible transcription factor. Our findings also suggest that the N-terminal fragment of pyrin is a potentially attractive target for anti-inflammatory drug discovery. During the next year, our objectives will be: (1) to elucidate the biochemical mechanism by which N-terminal pyrin activates NF-kappaB; (2) to extend studies in animal models, particularly a recently-developed line of pyrin-null mice; (3) to undertake structural studies of pyrin and related proteins; (4) to study gene expression profiles of patients with FMF during and between attacks; and (5) to undertake clinical trials of IL-1 inhibition in FMF and related disorders.